Perturbations in the genetic program of smooth muscle cell (SMC) differentiation underlie a variety of human diseases. While progress has been made in understanding the transcriptional regulation of a few SMC differentiation genes, one has been particularly recalcitrant to experimental analysis. This gene, the smooth muscle isoform of calponin (CNN1), is linked to a higher propensity for human malignancies, down-regulated in a number of vascular disorders, and dramatically up-regulated in SMC derived from cerebral blood vessels of Alzheimer's patients. The long-term goal of this lab is to elucidate the transcriptional circuitry governing CNN1 gene expression in both normal and pathological contexts (e.g., arterial disease). Towards this end, an integrated series of specific aims are proposed to address the hypothesis that CNN1 gene transcription requires the coordinate action of multiple regulatory modules containing SRF-binding CArG boxes that are under phenotype-dependent control. Aim 1 will exploit the recent procurement of bacterial artificial chromosome (BAG) transgenic mice and a new CNN1 knockout mouse to systematically evaluate CNN1 regulatory modules in vivo. Aim 2 endeavors to define human and mouse CNN1 expression in both in vitro and in vivo models of SMC phenotypic modulation and then assess DNA-protein associations by chromatin immunoprecipitation to gain a fundamental understanding of the CNN1 chromatin landscape under conditions in which CNN1 gene expression is altered. In Aim 3, SRF will be conditionally inactivated in SMC of mice with a highly specific Cre recombinase to assess unambiguously, and in a genetic manner, the in vivo requirement of SRF for CNN1 expression and the establishment of a transcriptionally competent chromatin landscape. The proposed studies will advance our understanding of the basic molecular underpinnings controlling CNN1 gene expression in vivo and hence the maintenance of a normal program of SMC differentiation. This in turn may provide fertile ground for the development of novel therapeutic interventions against a variety of devastating human diseases. Relevance of Research: Much of human disease is linked to the inappropriate turning on or off of genes. Our ability to control cardiovascular diseases and Alzheimer's for example, requires an understanding of how important genes in these diseases are turned on and off. This research proposal seeks to gain a deep understanding of how one such gene, called CNN1, is regulated under normal and disease-associated circumstances.